Brucellosis, Leptospirosis, Mycoplasmosis and Listeriosis are important zoonosis and represent an important cause of reproductive losses in animals worldwide, especially in Mediterranean countries and Egypt. Aiming at improvement in the diagnostic scheme, a quick method for the simultaneous detection of these microorganisms in different clinical samples using a SYBR green multiplex real-time polymerase chain reaction (m RT-PCR) has been developed. The m PCR has been standardized by using 4-pairs of primers to amplify 31kDa gene encoding protein in Brucella spp., lig gene in pathogenic Leptospira, hlyA gene in Listeria monocytogenes and 16S rDNA in Mycoplasma spp.  This study was applied to 161 different clinical samples (milk, blood, fetal fluids, semen, tissue and vaginal discharges). Real time PCR assay revealed a specific dissociation peak at Tm=79.0ºC, 80.0ºC, 85.2ºC and 88.0ºC (± 0.5) for Listeria, mycoplasma, brucella and Leptospira respectively. The real time assay neither revealed interferences between primers nor nonspecific florescence. In conclusion the high output, time saving and cost effective SYBR green m RT- PCR method established, could offer an effective tool for simultaneous, quick and reliable identification of these microbial agents in different clinical samples.